Research in the Alessi lab has been focused on understanding how mutations that truncate the C-terminal non-catalytic moiety of a protein kinases termed TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, spinocerebellar ataxia type 11 movement disorder.
In the course of the work Noor Esoof (PhD Student) and Ning Zhang (Postdoc) discovered that the Synaptic-vesicle-protein-2A (SV2A) which is a ubiquitous component of synaptic vesicles was efficiently phosphorylated by TTBK2 as well as related Casein kinase-1 family members.
Phosphorylation analysis indicated that TTBK2 and related kinases phosphorylated human SV2A at two constellations of residues; namely Cluster-1 (Ser42, Ser45 and Ser47) and Cluster-2 (Ser80, Ser81 and Thr84). Ning Zhang was also able to demonstrate that residues are also phosphorylated in endogenous SV2A in brain.
Excitingly, work carried out by Noor, Ning and Maximilian Fritsch (Postdoc) discovered that phosphorylation of Thr84 within Cluster-2 functions to trigger binding to the C2B domain of synaptotagmin-1, which is the key Ca2+ sensor for evoked synchronous neurotransmitter release at the synapse.
Working with Daan van Aalten, Maximilian Fritsch solved the crystal structure of the C2B domain of synaptotagmin-1 bound to an SV2A peptide phosphorylated at Thr84. This revealed that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326 and Lys328) on the surface of the synaptotagmin-1 C2B domain.
In a series of very elegant experiments undertaken by Michael Cousin and Sarah Gordon at the University of Edinburgh, they were able to demonstrate that synaptotagmin-1 retrieval during SV endocytosis was markedly dysfunctional when interaction of phosphorylated Thr84 with the C2B domain of synaptotagmin-1 was disrupted in primary cultures of mouse neurons.
This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1, and implicates SV2A as a phospho-dependent scaffold required for the specific retrieval of synaptotagmin-1 during synaptic vesicle endocytosis.
In future work it will be important to establish whether its TTBK2 or a related kinases that phosphorylates SV2A and whether disruption of this pathway is associated with spinocerebellar ataxia or any other movement disorder. It will also be important to understand the roles of cluster 1 and cluster 2 phosphorylation in greater detail. Knock-in mice in which phosphorylation sites have been ablated have been generated and these will be useful in enabling us to address this question.
To read the paper describing this work click here
In the course of the work Noor Esoof (PhD Student) and Ning Zhang (Postdoc) discovered that the Synaptic-vesicle-protein-2A (SV2A) which is a ubiquitous component of synaptic vesicles was efficiently phosphorylated by TTBK2 as well as related Casein kinase-1 family members.
Phosphorylation analysis indicated that TTBK2 and related kinases phosphorylated human SV2A at two constellations of residues; namely Cluster-1 (Ser42, Ser45 and Ser47) and Cluster-2 (Ser80, Ser81 and Thr84). Ning Zhang was also able to demonstrate that residues are also phosphorylated in endogenous SV2A in brain.
Excitingly, work carried out by Noor, Ning and Maximilian Fritsch (Postdoc) discovered that phosphorylation of Thr84 within Cluster-2 functions to trigger binding to the C2B domain of synaptotagmin-1, which is the key Ca2+ sensor for evoked synchronous neurotransmitter release at the synapse.
Working with Daan van Aalten, Maximilian Fritsch solved the crystal structure of the C2B domain of synaptotagmin-1 bound to an SV2A peptide phosphorylated at Thr84. This revealed that the phosphorylated Thr84 residue binds to a pocket formed by three conserved Lys residues (Lys314, Lys326 and Lys328) on the surface of the synaptotagmin-1 C2B domain.
In a series of very elegant experiments undertaken by Michael Cousin and Sarah Gordon at the University of Edinburgh, they were able to demonstrate that synaptotagmin-1 retrieval during SV endocytosis was markedly dysfunctional when interaction of phosphorylated Thr84 with the C2B domain of synaptotagmin-1 was disrupted in primary cultures of mouse neurons.
This study reveals fundamental details of how phosphorylation of Thr84 on SV2A controls its interaction with synaptotagmin-1, and implicates SV2A as a phospho-dependent scaffold required for the specific retrieval of synaptotagmin-1 during synaptic vesicle endocytosis.
In future work it will be important to establish whether its TTBK2 or a related kinases that phosphorylates SV2A and whether disruption of this pathway is associated with spinocerebellar ataxia or any other movement disorder. It will also be important to understand the roles of cluster 1 and cluster 2 phosphorylation in greater detail. Knock-in mice in which phosphorylation sites have been ablated have been generated and these will be useful in enabling us to address this question.
To read the paper describing this work click here